Efficient construction of gene targeting using phage-plasmid recombination

ABSTRACT

A method for producing gene targeting constructs in bacterial by way of homologous recombination between bacterial phage and plasmids.

BACKGROUND OF THE INVENTION

One of the most useful approaches for studying the functions of specificgenes (including their health related functions) is to examine theeffects of mutations within those genes (i.e., the phenotype of themutation). This approach involves correlating mutations within specificgenes with the phenotypes or disease conditions that result from thosemutations. This has been particularly fruitful in recent years with theidentification of genes for such diseases as cystic fibrosis (Snouwaertet al., Science, 257:1083 (1992)), obesity (Zhang et al, Nature, 372:425 (1994)), polycystic kidney disease (Moyer et al., Science, 264:1329(1994)), breast cancer [Miki et al., Science, 266:66-71 (1994);Tavtigian et al., Nat. Genet., 12:333-337 (1996)], and other diseases.In these cases, the function of the implicated genes was not apparentsolely from their DNA sequence but rather was defined by a diseasecondition associated with mutations in the genes.

A particularly productive approach to understanding the function of aparticular gene in animals involves the disruption of the gene'sfunction which is colloquially referred to as a "targeted mutagenesis".One common form of targeted mutagenesis involves generating "geneknockouts". Typically, a gene knockout involves disrupting a gene in thegermline of an animal at an early embryonic stage. (See, Thomas et al.,Cell, 51:503 (1987).) Once established in the germline, it is possibleto determine the effect of the mutation on the animal in both theheterozygous and homozygous states by appropriate breeding of micehaving the germline mutation.

Among the many examples of the use of knockout technology utilized toinvestigate gene function are U.S. Pat. Nos. 5,625,122 and 5,530,178 toMak, T. which describe the production of mice having a disrupted geneencoding lymphocyte-specific tyrosine kinase p56^(1ck) and Lyt-2,respectively. Silva et al., Science, 257:201 (1992) produced mice havinga disrupted α-Calcium Calmodulin kinase II gene (αCaMKII gene) whichresulted in animals having an abnormal fear response and aggressivebehavior. (See, also, Chen et al., Science, 266:291 [1994]). Wang etal., Science, 269:1108 (1995) demonstrated that the disruption in miceof the C/EPBα gene which encodes a basic leucine zipper transcriptionfactor results in impaired energy homeostasis in the mutant animals.Knudsen et al., Science, 270:960 (1995) demonstrated that disruption ofthe BAX gene in mice results in lymphoid hyperplasia and male germ celldeath.

The most common approach to producing knockout animals involves thedisruption of a target gene by inserting into the target gene (usuallyin embryonic stem cells), via homologous recombination, a DNA constructencoding a selectable marker gene flanked by DNA sequences homologous topart of the target gene. When properly designed, the DNA constructeffectively integrates into and disrupts the targeted gene therebypreventing expression of an active gene product encoded by that gene.

Homologous recombination involves recombination between two geneticelements (either extrachromosomally, intrachromosomally, or between anextrachromosomal element and a chromosomal locus) via homologous DNAsequences, which results in the physical exchange of DNA between thegenetic element. Homologous recombination is not limited to mammaliancells but also occurs in bacterial cells, yeast cells, in the slime moldDictyostelium discoideum and in other organisms. For a review ofhomologous recombination in mammalian cells, see Bollag et al., Ann.Rev. Genet., 23:199-225 (1989) (incorporated herein by reference). For areview of homologous recombination in fungal cells, see Orr-Weaver etal., Microbiol. Reviews, 49:33-58 (1985) incorporated herein byreference.

As is illustrated by the foregoing, gene knockout technology has oftenbeen used in mice and has allowed the identification of the function ofnumerous genes and, in some cases, ascertainment of their roles indisease. Much may be learned about the function of human genes fromstudies of mouse genetics because the vast majority of genes in humanshave homologous counterparts in the mouse. Because of this high level ofhomology between the species, it is now possible to define the functionof individual human genes and to elucidate their roles in health anddisease by making targeted germline mutations in selected genes in themouse. The phenotype of the resulting mutant mice can be used to helpdefine the phenotype in humans.

With the increasing awareness that mouse mutations can provide suchuseful insights about the function of genes from humans, a great deal ofinterest is developing to systematically generate mutations within genesin mice that correspond to those genes which are being isolated andcharacterized as part of various genome initiatives such as the HumanGenome Project. The problem with utilizing these procedures forlarge-scale mutagenesis experiments is that the technologies forgenerating transgenic animals and targeted mutations are currently verytedious, expensive, and labor intensive.

One of the biggest problems with the efficient generation of targetedmutations is the generation of the targeting construct. Targetingconstructs are typically prepared by isolating genomic clones containingthe region of interest, developing restriction maps, frequentlyengineering restriction sites into the clones, and manually cutting andpasting fragments to engineer the construct. See, e.g., Mak, T. U.S.Pat. Nos. 5,625,122 and 5,530,178; Joyner et al., Nature, 338:153-156(1989); Thomas et al., supra; Silva et al., supra, Chen et al., supra;Wang et al., supra; and Knudsen et al., supra. This process can take asingle highly skilled individual at least several weeks, often severalmonths, to complete. Thus, in order to more rapidly and efficientlyelucidate the functions of a variety of genes and to understand theirrole in health and disease, there exists a need to develop moreefficient methods for the production of targeting constructs which donot require detailed restriction mapping and certain other complexmolecular engineering steps.

SUMMARY OF THE INVENTION

The invention is directed to methods for producing gene targetingconstructs by way of homologous recombination between bacteriophage andplasmids. More particularly, the invention is directed to methods forproducing gene targeting constructs in bacteria by way of singlehomologous recombination events. The method comprises the steps ofpreparing a probe plasmid comprising a suppressor tRNA gene, preferablya bacterial replication origin and a probe DNA, the probe DNA comprisingat least a portion of a gene (e.g., exon) to be targeted. The probeplasmid is introduced into a population of homologous recombinationproficient, suppressor-free bacterial host cells.

The method further comprises preparing a target phage comprising atleast one suppressible mutation in a gene necessary for phage growth anda target DNA, the target DNA comprising a portion of a genomic region tobe targeted and which is homologous to all or part of the probe DNA ofthe probe plasmid. The population of bacterial cells containing theprobe plasmid is then infected with the target phage phage and probeplasmid are allowed to recombine via their homologous DNA. Recombinantphage may then be isolated by virtue of their ability to grow on asuppressor-free host cell by virtue of its incorporation of thesuppressor tRNA gene from the probe plasmid into the target phage.

Preferably, the probe DNA of the probe plasmids comprises at least about20 nucleotides to about 40 nucleotides of probe DNA. A preferredsuppressor tRNA gene for the practice of the present invention is SupF,an amber suppressor. The probe plasmid of the present invention mayfurther comprise a marker cassette, the marker cassette comprising thesuppressor tRNA gene and a mammalian cell selectable marker with themarker cassette being flanked on at least one side by probe DNA.Preferably, the marker cassette is flanked on both sides by at leastabout 20 nucleotides to 40 nucleotides or more of probe DNA. A preferredmammalian cell selectable marker for the practice of the presentinvention is the neo gene. However, other reporter genes such as thosethat confer antibiotic resistance to cells expressing the gene or markergenes which allow chemical or physical detection are also contemplatedas being within the scope of the present invention. The mammalian cellselectable marker is preferably, operatively linked to a promotercapable of allowing expression of the selectable marker gene inembryonic stem cells. A preferred promoter, according to the presentinvention, is the phophoglycerate kinase (PGK) promoter although otherpromoters which may function in embryonic stem cells are well known inthe art and fall within the scope of the present invention. Preferredbacteria phage for practice of the present invention is a lambda phage,although other bacteriophage may be used in the practice of theinvention.

The invention is also directed to methods for producing targetingconstructs by way of double recombination. The method comprises thesteps of preparing a circular probe plasmid/specific engineered fragment(SEF) comprising a marker cassette, the marker cassette comprising asuppressor tRNA gene and a mammalian selectable marker, the markercassette being flanked on each side by probe DNA homologous to a gene tobe targeted, and linker DNA, the linker DNA serving to link the probeDNA flanking the marker cassette so as to form a circular plasmid. Thecircular probe plasmid is then introduced into a population ofrecombination proficient suppressor-free bacterial host cells. Themethod further comprises preparing a target phage. The target phagecomprises at least one suppressible (e.g., amber) mutation in a genenecessary for phage growth and a target DNA comprising a portion of agenomic region to be targeted. The target DNA comprises DNA sequenceshomologous to all or part of probe DNA on the circular probe plasmid.The target phage is then used to infect the population of bacterialcells containing the probe plasmid/SEF and the phage and plasmid areallowed to recombine via their homologous DNA. The phage produced byhomologus recombination in the infected cells are then isolated, asdescribed above, for use as targeting constructs.

Preferably, the probe DNA of the circular probe plasmid each comprisesmore than about 40 bp of probe DNA. In a preferred embodiment S of thepresent invention, the suppressor tRNA gene is SupF. A preferredmammalian cell selectable marker for the practices of the invention is aneo gene, although other markers which confer, for example, antibioticresistance or which are chemically or physically detectable are alsocontemplated as falling within the scope of the present invention. Themammalian cell selectable markers are preferably operatively linked to apromoter capable of driving expression of the marker gene in anembryonic stem cell. A preferred promoter for the practice of theinvention is the phosphoglycerate kinase promoter (PGK), although otherpromoters which are capable of driving expression of a gene in embryonicstem cells are also within the scope of the invention. A preferred phagefor the practice of the present invention is a lambda phage.

The invention is also directed to targeting constructs produced by orobtainable by the methods of the present invention.

Another aspect of the present invention is directed to a method forproducing or obtaining targeting constructs comprising culturing undersuitable nutrient and environmental conditions a population ofhomologous recombination proficient bacterial cells comprising a targetphage of the invention and a probe plasmid or probe plasmid/SEF of thepresent invention and isolating the phage resulting from homologousrecombination between the target phage and the respective plasmids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts schematically the probe plasmid πANTg737-1.2 and Syrinx2A (Tg737-17).

FIG. 2 depicts schematically, homologous recombination between πAN13 anda target phage containing homologous target sequences and a strategy forthe single step selection for isolation of recombinant phage (targetingconstructs).

FIG. 3 shows the plasmid pBS/πAN/Neo used for double homologousrecombination.

FIG. 4 shows the SEF πANTg737 SEF-1.

FIG. 5 depicts recombination products resulting from single homologousrecombination and double homologous recombination.

FIG. 6 shows the SEF πTg737 SEF-2.

DETAILED DESCRIPTION

In one of its aspects, the present invention is directed to methods forproducing targeting constructs for the purpose of introducing into thegenome of an animal, a disruption at a particular genetic locus (i.e., atargeted mutation). The targeting constructs of the present inventionmay also be used to introduce into a genomic locus another functionalgene ("knock in") or to otherwise alter the function or expression of agene, for example, by knocking in a foreign promoter so as to place itin operative linkage with a gene in a chromosomal locus. The targetingconstruct is inserted into the appropriate genome location by takingadvantage of the cell's ability to mediate homologous recombinationbetween homologous sequences in the targeting construct and thesequences in the genomic region or gene of interest.

Unlike traditional methods for constructing targeting constructs, thepractice of the present invention does not require detailed restrictionmaps or extensive DNA sequence information in order to prepare targetingconstructs. Because such detailed information is not required to preparetargeting constructs according to the present invention, vectors may beproduced more quickly and effectively than previously employed methods.

More specifically, targeted mutagenesis of a gene refers to analteration (e.g., partial or complete inactivation) of normal productionor structure of the polypeptide encoded by the targeted gene of a singlecell, selected cells or all of the cells of an animal (or in culture) byintroducing an appropriate targeting construct into a site in the geneto be disrupted.

Targeted mutagenesis may also refer to "knocking in" a gene which meansreplacing one gene with all or part of another gene for the purpose ofdetermining, for example, whether two genes are functionally equivalent(see, e.g., Hanks et al., Science, 269:679 (1995), incorporated hereinby reference), although other applications are possible. For example,transcriptional regulatory sequences (elements) such as promoters may beknocked in to a region of a genome so as to become operatively linked toa structural gene thereby controlling expression of structural gene. Insome cases the transcriptional regulatory sequence may be knocked intoregions flanking the structural gene and yet still be positioned inoperation linkage with the gene.

In most cases, targeting constructs are constructed so as to include atleast a portion of a gene to be disrupted. Typically, the portion of thegene included in the targeting construct is interrupted by insertion ofa marker sequence (usually a selectable marker) that disrupts thereading frame of the interrupted gene so as to preclude expression of anactive gene product. This most often causes a knock out or inactivationof a gene. An exemplary selectable marker is the neo^(r) gene under thecontrol of a promoter that functions in the embryonic cells into whichthe marker is introduced. For example, the phosphoglycerate kinasepromoter (PGK) may be used to control expression of the neo gene therebyrendering the cells expressing the neo^(r) gene resistant to G418,although other promoters capable of driving expression of the selectablemarker in ES cells may also be used.

Prior to the present invention, the preparation of targeting constructstypically involved detailed restriction mapping in order to identifyconvenient restriction sites in the gene fragment to be used to "cut andpaste" DNA fragments to ultimately generate a targeting vector. However,mapping frequently reveals that convenient restriction sites are notavailable and therefore, they must be engineered into various componentsof the targeting constructs. According to the present invention,detailed mapping and sequence information are not required in order toprepare targeting constructs which results in a significant saving oftime and effort in preparing targeting constructs.

When such targeting constructs are introduced into embryonic stem cells,they can recombine with the target gene in the cell via the homologoussequences in both the construct and in the gene genomic region to bedisrupted. As discussed above, the result of the homologousrecombination event is often the insertion of a marker sequence into thetargeted gene, thereby disrupting the gene. Similarly, targetingconstructs designed for knocking in genes can recombine at thehomologous genomic site by homologous recombination and will result inthe introduction of all or a portion of a gene into that locus.Techniques for knocking in genes are described in detail in Hanks etal., Science, 269:679 (1995) which is incorporated herein by reference.

In order to introduce the targeting construct into the gemline of ananimal, the targeting construct is first introduced into anundifferentiated totipotent cell termed an embryonic stem (ES) cellwherein the construct can recombine with the selected genomic region viatheir homologous sequences. ES cells are derived from an embryo orblastocyst of the same species as the developing embryo into which theyare to be introduced. ES cells are typically selected for their abilityto integrate into the inner cell mass and contribute to the germ line ofan individual when introduced into the mammal in an embryo at theblastocyst stage of development. Thus, any ES cell line having thiscapability is suitable for use in the practice of the present invention.

The cells are cultured and prepared for introduction of the targetingconstruct using methods well known to the skilled artisan. (See, e.g.,Robertson, E. J. ed. "Teratocarcinomas and Embryonic Stem Cells, aPractical Approach", IRL Press, Washington D.C. [1987]; Bradley et al.,Current Topics in Devel. Biol. 20:357-371 [1986]; by Hogan et al. in"Manipulating the Mouse Embryo": A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor N.Y. [1986]; Thomas et al., Cell,51:503 [1987]; Koller et al., Proc. Natl. Acad. Sci. USA, 88:10730[1991]; Dorin et al., Transgenic Res., 1:101 [1992]; and Veis et al.,Cell, 75:229 [1993] all of which are incorporated herein by reference).The targeting construct may be introduced into ES cells by any one ofseveral methods known in the art including electroporation, calciumphosphate co-precipitation, retroviral infection, microinjection,lipofection and other methods. Insertion of the targeting construct intothe targeted gene is typically detected by selecting cells forexpression of the marker gene contained in the targeting constructwhich, as described above, is typically under the control of a promoterwhich is functional in the target cell type (i.e., promoters whichfunction in embryonic stem cells). ES cells expressing the markersequence are then isolated and expanded.

The ES cells having the disruption are then introduced into anearly-stage mouse embryo (e.g., blastocyst) (see, e.g., Robertson,supra, Bradley, supra, and Monsour et al., Nature, 336:348 (1988))incorporated herein by reference. Blastocysts and other early stageembryos used for this purpose are obtained by flushing the uterus ofpregnant animals for example, by the methods described in Robertson etal., supra and Bradley et al., supra. The suitable stage of developmentfor the blastocyst is species dependent, however, for mice it is about3.5 days post-fertilization.

While any embryo of the right age/stage of development is suitable forimplantation of the modified ES cell, preferred most embryos are maleand have genes coding for a coat color or other phenotypic marker thatis different from the coat color or other phenotypic marker encoded bythe ES cell genes. In this way, the offspring can be screened easily forthe presence of the targeted mutation by looking for mosaic coat color(e.g. agouti) or the other phenotypic markers (indicating that the EScell was incorporated into the developing embryo). Thus, for example, ifthe ES cell line carries the genes for white fur, the host embryosselected will preferably carry genes for black or agouti fur.

An alternate method of preparing an embryo containing ES cells thatpossess the targeting construct is to generate "aggregation chimeras". Amorula of the proper developmental stage (about 2 1/2 dayspost-fertilization for mice) is isolated. The zona pellucida can beremoved by treating the morula with a solution of mild acid for about 30seconds, thereby exposing the "clump" of cells that comprise the morula.Certain types of ES cells such as the R1 cell line for mice can then beco-cultured with the morula cells, forming an aggregation chimera embryoof morula and ES cells, (Joyner, A.L., "Gene Targeting", The PracticalApproach Series, JRL Press Oxford University Press, New York, 1993,incorporated herein by reference).

A refinement of the aggregation chimera embryo method can be used togenerate an embryo comprised of essentially only those ES cellscontaining the knockout construct. In this technique, a very early stagezygote (e.g., a two-cell stage zygote for mice) is given a mild electricshock. This shock serves to fuse the nuclei of the cells in the zygotethereby generating a single nucleus that has two-fold (or more) the DNAof a naturally occurring zygote of the same developmental stage. Thesezygotic cells are excluded from the developing embryo proper, andcontribute only to forming accessory embryonic structures such as theextra-embryonic membrane. Therefore, when ES cells are co-cultured withthe zygotic cells, the developing embryo is comprised exclusively of EScells, (see Joyner, A. L., supra).

After the ES cells have been incorporated into the aggregation chimeraor into the blastocyst, the embryos may be implanted into the uterus ofa pseudopregnant foster mother. While any foster mother may be used,preferred foster mothers are typically selected for their ability tobreed and reproduce well, and for their ability to care for their young.Such foster mothers are typically prepared by mating with vasectomizedmales of the same species. The pseudopregnant stage of the foster motheris important for successful implantation, and it is species dependent.For mice, this stage is about 2-3 days of pseudopregnancy.

Offspring that are born to the foster mother may be screened initiallyfor mosaic coat color or another phenotypic marker (where the phenotypeselection strategy has been employed). In addition, or as analternative, chromosomal DNA obtained from tail tissue of the offspringmay be screened for the presence of the targeted mutation using Southernblots and/or PCR. The offspring that are positive for homologousrecombination at the targeted locus will typically be a mosaic ofwild-type cells derived from the host embryo and heterozygous cellsderived from injected ES cells (i.e., chimeric offspring). Chimericoffspring are crossed with wild-type partners to generate offspring thatare heterozygous for the targeted mutations, i.e., all of their cellsare heterozygous for the mutation.

Methods for producing transgenic mammals, including rabbits, pigs, andrats, using micro-injection are described in Hamer et al., Nature315:680-683 (1985).

If animals homozygous for the targeted mutation are desired, they can beprepared by crossing animals heterozygous for the targeted mutation.Mammals homozygous for the disruption may be identified by Southernblotting of equivalent amounts of genomic DNA from mammals that are theproduct of this cross, as well as mammals of the same species that areknown heterozygotes, and wild-type mammals. Alternatively, specificrestriction fragment length polymorphisms can be detected whichco-segregate with the mutant locus. Probes to screen the Southern blotsfor the presence of the targeting construct in the genomic DNA can bedesigned as described below.

Other means of identifying and characterizing the offspring having adisrupted gene are also available. For example, Northern blots can beused to probe mRNA obtained from various tissues of the offspring forthe presence or absence of transcripts. Differences in the length of thetranscripts encoded by the targeted gene can also be detected. Inaddition, Western blots can be used to assess the level of expression ofthe targeted gene by probing the Western blot with an antibody againstthe protein encoded by the targeted gene. Protein for the Western blotmay be isolated from tissues where this gene is normally expressed.Finally, in situ analysis (such as fixing the cells and labeling withantibody or nucleic acid probe) and/or FACS (fluorescence activated cellsorting) analysis of various cells from the offspring can be conductedusing suitable antibodies to look for the presence or absence of thegene product.

While the foregoing discussion describes the use of targeting constructsto introduce DNA into a genomic locus via homologous recombination, theprocess of homologous recombination may also, according to the presentinvention, be used to prepare the targeting constructs themselves.

The methods of the present invention exploit certain aspects of the πvxscreening procedure described by Seed (Nucleic Acid Res. 11:2427-2445,1980) and allows the rapid generation of target vectors for theintroduction of mutations into mice, without the necessity of a detailedrestriction map and with limited DNA sequence information. The methodsof the present invention take advantage of the ability of DNA sequencesin a plasmid to recombine with homologous DNA in a bacteriophage("phage") with the resulting insertion of at least a portion ofplasmid-born DNA into the phage.

By way of overview, the invention is directed to a method for preparingtargeting constructs by preparing a "probe" plasmid construct comprisingDNA homologous to a gene to be targeted (probe DNA), a suppressor t-RNAgene and preferably a gene encoding a mammalian selectable marker. Atarget phage having DNA comprising at least a portion of a gene to bedisrupted and which has homology to the DNA in the "probe" plasmid andone or more suppressible mutations in genes important for growth of thephage is also prepared. The probe plasmid is then introduced into apopulation of recombination competent bacterial cells. The population ofcells containing the probe plasmid is then infected with the targetphage. The target phage and the probe plasmid can then recombine viatheir aforementioned homologous DNAs which results in the insertion ofprobe plasmid sequences into the homologous region of the phage insertvia their homologous sequences. The recombined phage can then beisolated, amplified and used as a targeting construct for mammaliancells.

In another aspect of the invention, a probe plasmid is prepared in which"probe" DNA comprising a portion of a cDNA or genomic DNA to be targetedare inserted into a small plasmid containing a suppressor t-RNA gene.Exemplary plasmids include πVX (Seed, supra), πAN13 (Sambrook et al.,Molecular Cloning: A Laboratory Manual, section p. 1.19, Cold SpringHarbor Laboratories (1989)), and πAN7 (Lutz et al., Proc. Natl. Acad.Sci. USA, 84:4379 (1987) and Ausubel et al., p. 5.0.1-5.11.2(incorporated herein by reference)). Methods for producing cDNAlibraries from which to obtain the relevant cDNA are well known in theart and are described in Sambrook et al., p. 8.2-8.93 (incorporatedherein by reference). The probe plasmid may also comprise a marker forselection in mammalian cells (e.g., embryonic stem cells). The mammalianmarker may allow biochemical selection, e.g., by conferring resistanceto an antibiotic, e.g., or physical selection, e.g., expression of afluorescent protein luciferase, or other markers whose expression may bedetected by physical means. Preferably, the selectable marker is flankedon each end by probe DNA. Such flanking probe DNA is preferably at leastabout 20 to about 40 bp in length. The probe plasmid is then introducedinto recombination-proficient bacterial cells (suppressor-less) by, forexample, electroporation. Other methods for introducing plasmids intocells are described in Sambrook et al., supra and are well known in theart. Probe plasmids for use in preparing targeting constructs forknocking in promoter sequences, for example, comprise a marker cassettein which the selectable marker and the suppressor t-RNA gene are in 5'juxtaposition to the DNA to be knocked in. Alternatively, probe plasmidscalled specific engineered fragments (SEF) may be constructed whichcomprise the selectable markers and 40 or more base pairs of probe DNAflanking each side of a spacer or linker DNA. Such SEFs are useful forgenerating target constructs by double homologous recombination.

The population of bacterial cells containing the probe plasmid is theninfected with target phage containing the genomic site to be targeted.An amber mutation in a gene or genes of the phage that regulate lyticgrowth is important. The target phage may be obtained from a genomiclibrary prepared by methods well known in the art and described indetail in Sambrook et al., p. 9.2-9.58 or Ausubel et al., p.5.0.1-5.11.2, both of which are incorporated herein by reference.Preferably, the vector phage also has an amber suppressible mutation ina gene which, when the mutation suppressed, is readily detectable, e.g.,genes for lytic growth or an lacZ encoding β-galactosidase.

Cells containing the probe plasmid are then infected with a target phagesuch as those described above, after which the probe plasmid recombineswith the target phage by homologous recombination between theirhomologous sequences. Only those phage that incorporate the gene viahomologous recombination with the probe plasmid will be able to grow onsuppressor-free host cells and can thus be readily isolated. Insertionof plasmid sequences including the selectable marker into phageinterrupts the gene sequence carried by the target phage.

DNA isolated from these newly generated phage can then be used astargeting constructs to introduce targeted mutations, for example, in EScells using methods described above. The examples set out below use themouse Tg737 gene as a model to test how efficiently the targetingconstructs could be generated by homologous recombination in bacteria.

Example 1 describes the production of targeting constructs by singlehomologous recombination.

Example 2 describes the production of targeting vectors by doublehomologous recombination using probe plasmids referred to as specificengineered fragments.

The approach is illustrated with reference to particular plasmids andparticular phage. However, other plasmids having similar characteristicsincluding suppressor genes and the ability to carry fragments of genomicor cDNA and other appropriate selectable markers may be used. Similarly,different phage may also be used so long as it contains appropriatesuppressible mutations which may be complemented by the t-RNA suppressorgenes provided by the plasmid.

EXAMPLE 1 Generation of Targeting Vectors by Single HomologousRecombination in Bacterial Host Cells

A model system was used to demonstrate that targeting vector can beprepared by taking advantage of the fact that homologous recombinationcan occur between target phage and probe plasmids in bacterial cells. Asa model gene, the mouse Tg737 was utilized (Moyer et al., Science,264:1329 (1994) incorporated herein by reference).

To generate a target phage comprising a lambda phage bearing ambermutations, a 17 kb SalI insert of a wild-type genomic clone containing aportion of the mouse Tg737 gene (including exon II of the genes),(target gene) was subcloned into Syrinx 2A phage arms (Lutz et al.,Proc. Natl. Acad. Sci. USA, 84:4379 [1987] incorporated herein byreference) and the phage was packaged using methods well known in theart (see FIG. 1). Although a 17 kb portion of the Tg737 gene was used inthis example, the size of the gene used in the target phage may vary.Preferred sizes are from about 15 kb-20 kb. Further, the insert need notbe a portion of a genomic DNA, but may also be a portion of a cDNA.Syrinx 2A phage, a lambda phage vector, carries multiple cloning sitesand a rap gene, which is required for efficient phage-plasmidrecombination. The Syrinx 2A phage has amber mutations in the lambda A,B, and S genes which are essential for lytic growth and therefore, thefrequency of spontaneous reversion is very low. The resulting phagecarrying the target Tg737 DNA is called Syrinx 2A (Tg737-17).

In order to prepare a probe plasmid, a 1.2 kb EcoRI genomic fragment ofthe Tg737 gene including the ATG containing exon II of Tg737 (probe DNA)was isolated from the cloned genomic sequences and inserted in the πAN13plasmid (see Sambrook et al., supra and Lutz et al., supra) to generatethe probe plasmid πANTg737-1.2 (see FIG. 1); stretches of sequence asshort as 20-40 bp can also be used for these experiments. Probe DNA mayalso be derived from a cDNA as well as a genomic DNA. The πAN13 plasmidis a high copy number miniplasmid which is designed for efficientrecombination screening and includes a supF gene capable of producing asuppressor t-RNA capable of suppressing the amber mutation found in theSyrinx 2A (Tg737-1.2).

Plasmid πANTg737 plasmid was then introduced into E. coli MC1061 (sup0)by electroporation (see Sambrook et al., p. 1.74-1.75). The resultingpopulation of E. coli containing πANTg737-1.2 was then infected withSyrinx 2A (Tg737-17) using routine methods (see Sambrook et al., p. 2.6et seq.) and homologous recombination was allowed to occur betweenSyrinx 2A (Tg737-17) and πANTg737-1.2

As depicted in FIG. 2, homologous recombination between the target phage[Syrinx 2A(Tg737-17)] and the probe plasmid (πANTg737-1.2) results inthe integration of the supF gene into Tg737 gene segment of the targetphage via its homologous Tg737 sequences and in the duplication of theprobe DNA. The integration of supF gene into the recombinant phage wasestablished by the production of active β-galactosidase in LG75 cells(sup)0, LacZAM).

Integration of the probe DNA into homologous DNA in the target phageoccurs by what is referred to as single homologous recombination and, asdescribed above, results in the duplication of the probe plasmidsequences at the recombination site in the target phage. When thetargeting construct having the duplication is introduced into the targetgenomic site in mammalian cells, the presence of such duplicatedsequence may allow alternative splicing to occur after transcription ofthe disrupted gene thereby splicing out the disruption. However, thepossibility of such alternative splicing may be minimized by preparingprobe plasmids having a genomic or cDNA fragment of at least 20-40 bpthat occur within the open reading frame of a single exon. This willcause a duplication of a part of the exon. The reading frame will shiftand cause a disruption in gene expression if the size of the duplicatedregion is chosen carefully (i.e., not a multiple of 3 bp).

A simple one-step genetic selection was developed for detection of phagewhich have undergone homologous recombination with probe plasmid and isillustrated schematically in FIG. 2. Two criteria were used to establishthat true homologous recombination had occurred.

First, integration of supF gene carried by the probe plasmid into targetphage carried by the target phage generates recombinant phage which cangrow in suppressor-less (or Sup°) host cells and by suppressing theamber mutation in genes for lytic growth and in the β-galactosidase ofthe phage thereby allowing the production of active β-galactosidase.These phage are easily detected by their ability to give rise to blueplaques when plated on suppressor-less, LacZAM cells such as E. coliLG75.

Second, homologous recombination is a reversible process. The frequencyof reversal of homologous recombination event has been estimated to beon the order of 10² to 10³ per generation. The frequency of excision ofplasmid sequences was detected by propagation of recombinant phage inrecombination-proficient host cells. The phages that have lost the SupFgene can only grow in supF⁺ (E. coli LE392) host cells but not in SupF-host cells (LG75).

Finally, homologous recombination was finally confirmed by restrictionmapping using EcoRI, BamHI, SalI, HindIII, XbaI or SacI. Based on thisanalysis and the known restriction map of πAN13 were shown to arise fromhomologous recombination between the target phage Syrinx 2A (Tg737-17)and the probe plasmid πAN13 (Tg737-1.2).

These studies establish that targeting constructs can be efficientlygenerated by single homologous recombination in bacteria according tothe methods of the present invention, without the extensive DNA sequenceinformation and without the detailed restriction mapping normallyrequired by standard methods.

EXAMPLE 2 Double Homologous Recombination in Bacteria

As discussed above, targeting constructs generated by single homologousrecombination have a duplication of sequences (portion of genomic orcDNA corresponding to region to be disrupted) at the insertion site inthe target phage. However, it is possible to generate probe plasmidswhich are used for generating targeting constructs by double homologousrecombination which do not result in duplicated target sequences.

By way of example, a plasmid, pBS/πAN/neo, which contains the SupF geneand neo^(r) gene is constructed as outlined in FIG. 4 by inserting aSupF gene, a colE1 gene and a neo^(r) gene into the multiple cloningsite of the plasmid pBluescript (Stratagene, La Jolla, Calif.). Thisplasmid is used as template to produce a plasmid/specific engineeredfragment using chimeric primers in a PCR reaction. The chimeric primerspreferably contain more than 40 bases of sequence corresponding to the5' and 3' ends of exon II of the Tg737 gene and preferably 20 bases ofsequences corresponding to the 5' of the SupF gene and the 3' end of thefunctional neo^(r) gene of pBS/πAN/neo. Exemplary primers include,

    ______________________________________                                        CAAATGATGGAAAATGTTCATCTGGCACCAGAAACAGATG                                      (SEQ ID NO: 1)                                                                CTCAGTATCATAGGCTGGGTTGTAGTCGTTGAAACCAGAG                                      (SEQ ID NO: 2)                                                                ______________________________________                                    

Circularization of the 2.5 kb PCR generated fragment is achieved bycloning in a T/A vector or by ligation with synthetic linker and therebygenerating a plasmid, πTg737SEF1 (Tg737SEF1).

The plasmid Tg737 SEF1 is then introduced into E. coli strain[Mc1061cp3] and the bacterial cells containing Tg737SEF1 are theninfected with Syrinx 2A (Tg737-17) to recombine with the phage asdescribed in Example 1. Recombinant phage are then selected according tothe method set out in Example 1. The phage selected with this procedurecan result from both single and double homologous recombination.Selection of recombinant phage resulting from double homologousrecombination is accomplished by a scheme outlined in FIG. 5. Briefly,as illustrated in FIG. 5, recombinant phage may be grown undernon-selective conditions. Phage having undergone a single homologousrecombination event and the resulting duplication of probe sequenceswill undergo a spontaneous excision of the duplicated sequences whichalso results in the loss of the SupF gene. Phage are then plated onsuppressor-less host cells wherein only the phage having undergone thedouble homologous recombination event will retain the SupF gene and itsflanking probe DNA and will grow on the suppressorless cells.

For selection of double homologous recombination, a large plasmid,πTg737SEF2, derived from πTg737SEF1 is constructed by inserting the 8.6kb BamHI fragment of the "stuffer" region of Syrinx 2A into the uniqueBamHI site of πTg737SEF1 (FIG. 9). The integration of the πTg737SEF2plasmid into the target phage can occur by either single homologousrecombination, in which case the entire plasmid will integrate, or bydouble homologous recombination, where only the SupF-neo cassette willintegrate into the target via the homologous. The single homologousrecombination event will be selected against because the integratedplasmid will make a phage genome too large to package into a singlephage head. Therefore only the double homologous recombination eventwill be selected for.

As discussed in detail above, the foregoing examples demonstrate thattargeting constructs may be produced efficiently, according to thepresent invention. Without detailed DNA sequence information orrestriction mapping, thereby eliminating a critical bottle-neck in thegeneration of targeted mutations in amber. The foregoing examples arepresented by way of illustration and are not intended to limit the scopeof the invention as set out in the claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 2                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #    40            TTCA TCTGGCACCA GAAACAGATG                                 - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 40 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #    40            GGGT TGTAGTCGTT GAAACCAGAG                                 __________________________________________________________________________

What is claimed is:
 1. A method for producing targeting constructs bydouble homologous recombination, the method comprising the steps of:a)preparing a circular probe plasmid comprising a marker cassette and saidmarker cassette comprising a suppressor t-RNA gene, a mammalian cellselectable marker, said marker cassette flanked on each side by probeDNA homologous to a gene to be targeted and a linker DNA, said linkerDNA linking the probe DNA flanking said marker cassette so as to form acircular plasmid; b) introducing the probe plasmid of step 1 into apopulation of recombination proficient suppressor-free bacterial hostcells; c) preparing a target phage, said target phage comprising atleast one suppressible mutation in a gene necessary for phage growth,and a target DNA comprising a portion of a genomic region to be targetedand wherein said target sequence is homologous to all or part of theprobe DNA of step a); d) infecting the population of bacterial cells ofstep b) with the bacteriophage of step c), thereby allowingrecombination between the probe DNA and the target DNA; e) isolatingphage produced in step d).
 2. The method of claim 1 wherein said probeDNA comprises from about at least 40 to about 100 nucleotides.
 3. Themethod of claim 1 wherein said probe DNA each comprise from about atleast 20 to about 40 nucleotides.
 4. The method of claim 1 wherein saidsuppressor t-RNA gene is SupF.
 5. The method of claim 1 wherein themammalian cell selectable marker is a neo^(r) gene.
 6. The method ofclaim 4 wherein said neo gene is operatively linked to a promotercapable of directing expression of the neo gene in embryonic stem cells.7. The method of claim 5 wherein the promoter is the phosphoglyceratekinase promoter.
 8. The method of claim 1 wherein the phage is a lambdaphage.
 9. The method of claim 1 wherein the suppressible mutations areamber mutations.